Models are necessary to study certain disease characteristics. In infectious disease research and vaccine development, it is important to study the cellular and humoral immune response, both together and separately. A 2006 report by the World Health Organization stated “There is evidence suggesting that both cellular and humoral immune responses contribute to protection in humans, and thus the development of laboratory methods and animal models for evaluation of new acellular pertussis vaccines/combinations which detect both types of immune response is necessary.”
Mice are the most commonly used research model animal as they are easy to maintain, reproduce readily, and are less costly to house and care for than large animal models. In addition, the ability to readily genetically modify the mouse genome has enabled the creation of many mouse lines with specific phenotypes that are useful for research (Malakoff, 2000). There are numerous existing immune-deficient mouse models.
Immune-deficient mice with spontaneous mutations were first characterized in 1966-68 (nude) and 1983 (scid). The nude mutation causes a failure of most T-cells to develop from their thymic precursor cells although some T-cells, B-cells and NK cells are present. Therefore the nude mouse is not an immunologically inert mouse although it has been used extensively in research. Mice homozygous for the mutation scid (SCID mice) have no B or T lymphocytes, however they do have NK cells. In addition, they often have a “leaky” phenotype in which immunoglobulins are produced (Bosma et al. (1989) Curr Top Microbiol Immunol 152, 1-263, Bosma & Carroll, (1991) Ann. Rev. Immunol. 9, 323-350). A Rag-1 or Rag-2 deficient mouse has also been developed (Mombaerts et al., (1992) Cell 68, 869-877, Shinkai, et al. (1992) Cell 68, 855-867, U.S. Pat. No. 5,859,307). Rag-1 and Rag-2 deficient mice cannot initiate V(D)J rearrangement and therefore lack mature lymphocytes. SCID mutant and Rag-1 and Rag-2 null mice have been widely used in studies with T-cell deficient nude mice in preconditioning, transplantation, and pathogen challenge protocols.
One way the body reacts to antigens is by making antibodies. Antibodies (also called immunoglobulins or “Igs”) are proteins that are manufactured by B-cells and circulate in the blood to detect foreign antigens When a vertebrate first encounters an antigen, it exhibits a primary humoral immune response, where B lymphocytes are activated, which generate highly specific antibodies to the antigen and differentiate into “effector” cells to secrete the antibodies. If the animal encounters the same antigen again after a short time the immune response is more rapid and has a greater magnitude than the first response. The initial encounter causes certain B-cells to proliferate and differentiate. The progeny lymphocytes include not only effector cells but also memory cells, which retain the capacity to produce effector cells upon subsequent stimulation by the original antigen. The effector cells live for only a few days but memory cells will remain active for an extended period, even throughout the animal's life, and can be reactivated by a second stimulation with the same antigen. Thus, when an antigen is encountered again, the memory cells quickly produce effector cells, which rapidly produce antibodies.
To examine the role of B-cells in immune responses, mice have been made B-cell deficient by antibody depletion (Gordon, 1979), T-cell reconstitution of SCID mice (Ronchese and Hausmann, 1993; Sunshine et al., (1991) J Exp Med 174, 1653-1656), or by genetic disruption of the Ig locus (Kitamura et al., (1991) Nature 350, 423-426; Jakobovits et al., (1993) Proceedings of the National Academy of Sciences of the United States of America 90, 2551-2555; Chen et al., (1993) Int Immunol 5, 647-656).
The use of B-cell deficient mice, in side by side studies with wild type mice, SCID, Rag-1 or Rag-2 null mice, has allowed T-cell (cellular) responses to be studied separately from B-cell (humoral) responses for many diseases. For example, immune responses by B-cell deficient mice have been studied for the bacterial diseases Salmonella, Bodetella, and Tularemia (Ugrinovic et al., (2003) Infect Immun 71, 6808-6819, Leef et al., (2000) J Exp Med 191, 1841-1852, Chen et al., (2004) Microbial pathogenesis 36, 311-318.), the viral disease Smallpox (Wyatt et al., (2004) Proceedings of the National Academy of Sciences of the United States of America 101, 4590-4595), the parasitic diseases Leishmania (Brown and Reiner, (1999) Infect Immun 67, 266-270, Miles et al., (2005) J Exp Med 201, 747-754, Ronet et al., (2008) J Immunol 180, 4825-4835.) and Malaria (Weidanz et al., (2005) Exp Parasitol 111, 97-104), and for inflammatory bowel disease (Ma et al., (1995) J Exp Med 182, 1567-1572). In fact, papers detailing production of three different lines of Hc KO, B-cell deficient mice produced in the 1990's (Kitamura et al., (1991) Nature 350, 423-426; Chen et al., (1993) Int Immunol 5, 647-656; Jakobovits et al., (1993) Proceedings of the National Academy of Sciences of the United States of America 90, 2551-2555) have to date been cited by over 1000 scientific publications (source: Google Scholar search engine). In addition to studying the basic immune response to a pathogenic challenge, these animals have also been used to test vaccines for effectiveness (Wyatt et al., (2004) Proceedings of the National Academy of Sciences of the United States of America 101, 4590-4595). The use of B-cell deficient transgenic mice also allowed for the determination of the critical role for B-cells in the systemic autoimmunity phenotype associated with lupus (Chan et al., (1999) Immunol Rev 169, 107-121).
Although mice are used extensively, there are few examples in the literature for the use of other species as immuno-deficient animal models for human disease research. Most of these are very specialized in relation to the animal chosen and the disease being modeled. Rats have been immunosuppressed by administration of dexamethasone prior to infection with aspergillus to test the efficacy of prophylactic drugs (Ulmann et al., (2007) J Antimicrob Chemother 60, 1080-1084). A ligated germ-free rabbit appendix was utilized to study inflammatory responses related to inflammatory bowel disease (Shanmugam et al., (2005) Inflamm Bowel Dis 11, 992-996).
PCT Publication No. WO06/047603 and related U.S. Patent publications U.S. 2006/0130157 and U.S. 2008/0026457, which are incorporated in their entirety herein, describe certain ungulate immunoglobin germline gene sequence arrangement as well as genomic sequences encoding the heavy chain locus of ungulate immunoglobulin, and ungulate cells, tissues and animals that lack an allele of a native heavy or light chain immunoglobulin gene and specifically describe methods of targeted disruption of individual porcine Ig gene sequences as well as replacement with human Ig genes. The resulting genetically modified animals are described as useful to produce human polyclonal antibodies as therapeutics.
Mice have been genetically modified to make them more appropriate models for infection with human pathogens. For example, species-specific ligand/receptors exist for infections such as Listeria. These receptors have been introduced as transgenes into mice. These transgenic mice then express the receptor and become more susceptible hosts as the pathogen can bind to its known receptor (which is not present in wild type mice) and initiate infection (see Lecuit and Cossart et al., (2002) Trends Mol Med 8, 537-542). Although such mouse models have proven useful, researchers still stress that there are technical limitations, as animals which normally develop listeriosis are not classical laboratory animals such as the rat or mouse, but rather farm animals (Lecuit, (2007) Microbes Infect 9, 1216-1225).
There remains a need for an animal system that can be effectively used to model human disorders, and in particular to model immune deficiencies. There also remains a need for an effective animal model to develop novel therapeutics. There further remains a need for a large animal model to develop and produce proteins, in particular human immunoglobulins, without endogenous contamination.
It is an object of the invention to provide improved animal systems to model human disorders and to allow production of certain therapeutic agents, particularly certain biologics.